Project Summary/Abstract Epigenetic regulatory pathways governing gene expression are intimately involved in the regulation of early heart development and cardiac remodeling under pathological stress. Disrupting the cardiac transcriptional networks during early heart development and cardiac regeneration may lead to heart diseases. Among all the known epigenomic modifiers, the Ten-Eleven Translocation (TET) protein family is a relatively new member found to mediate the reversal of DNA methylation in the mammalian genome. The TET dioxygenases (TET1-3) are capable of converting 5-methylcytosine (5mC) to 5-hydroxymethyl-cytosine (5hmC) and further oxidized species, thereby promoting active DNA demethylation. The dynamic changes in 5mC/ 5hmC distributions and transcriptional reprogramming play vital roles during early CM development, a critical period that also provides an optimal time window to study fundamental epigenetic regulatory mechanisms that govern cardiac gene transcription. Our own preliminary studies revealed that genetic depletion of Tet proteins in mice impaired early cardiomyocyte (CM) development. At the cellular level, Tet-deficient CMs further exhibited reduced proliferation and metabolic dysfunction. At the molecular level, upon Tet deletion, we observed massive changes in DNA methylation and a disorganized chromatin architecture that might account for disrupted cardiac transcriptional networks and abnormal expression of key metabolic genes involved in proliferation, glycolysis and mitochondrial respiration in CMs. We hypothesize that the Tet-mediated DNA demethylation pathway is critical for maintaining proper chromatin accessibility and chromatin looping, thereby regulating transcriptional programming to instruct CM development. The immediate availability of a cardiac-specific Tet triple knockout mouse model, as well as a set of innovative tools developed for precise mapping and editing of DNA modifications, has placed us in an extremely competitive position to unravel novel epigenetic regulatory mechanisms controlling CM development. In Aim 1, we will define how Tet/5hmC regulate chromatin accessibility and the binding of key transcriptional factors to their targets to program essential transcriptional outputs and maintain proper CM development. In Aim 2, we will examine how Tet/5hmC regulate chromatin looping by interplaying with enhancer and insulator elements at critical genomic loci to control metabolic gene expression during CM development. Upon completion of our proposed studies, we anticipate to establish a new paradigm by introducing a previously underappreciated dimension in the epigenetic regulation of the cardiovascular system. Findings from our proposed studies will also provide novel insights into the molecular mechanisms responsible for cardiac gene transcription and heart development, thereby forming a solid basis for developing potential epigenetic therapies to prevent and treat congenital heart diseases.